This invention relates to methods for converting compactin to pravastatin using an agent derived from the filamentous bacterium Actinomadura, to methods for lowering cholesterol levels in mammals, and to Actinomadura and to Actinomadura hydroxylase.
One of the major causes of atherosclerosis and coronary disease is attributed to high blood cholesterol levels. It has been estimated that at least about 50% of total body cholesterol is derived from de novo cholesterol synthesis. A major rate-limiting step in the cholesterol biosynthetic pathway is catalyzed by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase. Compactin and pravastatin have been reported to be competitive inhibitors of HMG-CoA reductase, and the presence of either one can result in inhibition of cholesterol biosynthesis.
Microbial hydroxylation of compactin can produce hydroxylated forms of compactin, e.g., pravastatin. Some hydroxylated forms are reportedly more effective than compactin as competitive inhibitors of HMG-CoA. It has been reported that this hydroxylation can be effected to differing degrees by many different genera of fungi, and from the bacteria Nocardia and Streptomyces roseochromogenus and Streptomyces carbophilus. See, e.g., U.S. Pat. No. 5,179,013; U.S. Pat. No. 4,448,979; U.S. Pat. No. 4,346,227; U.S. Pat. No. 4,537,859; Canadian Patent No. 1,150,170; Canadian Patent No. 1,186,647; Serizawa et al., J. Antibiotics 36:887-891 (1983).
A problem with using fungi for the production of pravastatin is that they generally do not tolerate increases in the amount of compactin added to the culture medium, presumably due to the anti-fungal activity of compactin. Serizawa et al., J. Antibiotics 36:887-891 (1983).
The cytochrome P450 system has been shown to be required for the hydroxylation of compactin to pravastatin in Streptomyces carbophilus. Matsuoka et al., Eur. J. Biochem. 184:707-713 (1989). Problems with the use of such an enzyme is that it is a complex of proteins rather than a single protein, making recombinant DNA manipulations difficult, and that compactin, which is an inducer of the cytochrome P450 system, is very expensive.
It is an object of the invention to provide an effective and relatively inexpensive method for converting compactin to pravastatin.
It is another object of the invention to use Actinomadura, a filamentous bacterium, to convert compactin to pravastatin.
It is yet another object of the invention to use an Actinomadura hydroxylase to convert compactin to pravastatin.
It is yet another object of the invention to use an Actinomadura constitutive hydroxylase that does not require compactin as an inducer, to convert compactin to pravastatin.
Still another object of the invention is to use pravastatin, derived from an Actinomadura hydroxylase that converts compactin to pravastatin, to treat a mammal so as to lower the mammal""s blood cholesterol level.
According to the invention, a method for converting compactin to pravastatin is provided. Compactin is provided and contacted with an agent, e.g., a hydroxylation enzyme, e.g., a hydroxylase that is, e.g., constitutive and cytochrome P450 system-independent, derived from Actinomadura, under conditions in which the agent converts compactin to pravastatin. In certain embodiments, the pravastatin is isolated.
Preferably, the compactin is provided by providing, e.g., a microorganism, e.g., a fungus or bacterium, that produces compactin, or a cell free extract of a microorganism that produces compactin, or cell free culture media from a pregrown culture of a microorganism that produces compactin, or a solution comprising compactin, or semi-purified compactin, or substantially purified compactin.
In certain embodiments, the compactin is contacted with the agent, e.g., by contacting whole cells of Actinomadura with the compactin, or by contacting a cell free extract of Actinomadura with the compactin, or by contacting cell free culture media from a pregrown culture of Actinomadura with the compactin, or by contacting a solution having the Actinomadura agent with the compactin, or by contacting semi-purified or substantially purified Actinomadura agent with the compactin.
Variations include, e.g., contacting, e.g., a culture, e.g., a pregrown culture or a starting culture, or a cell free extract, of the microorganism that produces compactin , or semi-purified or substantially purified compactin, with, e.g., a culture, e.g., a pregrown culture or a starting culture, or a cell free extract, of Actinomadura, or semi-purified or substantially purified Actinomadura agent.
Another aspect of the invention is a cell free extract derived from Actinomadura, having an agent, e.g., a hydroxylase, that converts compactin to pravastatin.
Another aspect of the invention is a hydroxylase, e.g., semi-purified or substantially purified, from Actinomadura that converts compactin to pravastatin, wherein the hydroxylase is a constitutive enzyme, wherein the activity of the hydroxylase is stimulated by any of ATP, ascorbic acid or Mg++, but not by Fe++ or Fe+++, and wherein the hydroxylase is cytochrome P450 system-independent.
Another aspect of the invention is purified Actinomadura ATCC 55678 having an agent for converting compactin to pravastatin.
Yet another aspect of the invention is a method for treating a mammal to lower the blood cholesterol level of the mammal. Pravastatin, derived from compactin by contacting the compactin with an agent, e.g., a hydroxylase, derived from Actinomadura that converts compactin to pravastatin, is provided. The pravastatin is administered to a mammal in need of such treatment to cause a lower blood cholesterol level in the mammal.
The above and other objects, features and advantages of the present invention will be better understood from the following specification.
This invention provides a method for converting compactin to pravastatin. Compactin is provided and contacted with an agent derived from Actinomadura under conditions in which the agent converts compactin to pravastatin. In certain embodiments, the pravastatin is isolated.
Compactin (also known as mevastatin, ML-236B, and CS-500) is meant to include, e.g., the acid form (also known as ML-236B carboxylic acid), the lactone form (also known as ML-236B lactone), and salts and esters thereof. The lactone form of compactin may be represented by the formula (I): 
A preferred compactin is the sodium salt of compactin.
Pravastatin (also known as eptastatin, mezalotin, pravachol, CS-514, and SQ-31000) is meant to include, e.g., the acid form, the lactone form, and salts and esters thereof. The 3xcex2-hydroxy lactone form of pravastatin may be represented by the formula (II): 
Other forms of hydroxylated compactin include compounds in which the hydroxyl group is added at other positions of the compactin molecule, e.g. at position 6.
The compactin can be provided in any way which enables the agent to act upon it. For example, compactin can be provided by providing a microorganism, e.g., a fungus or bacterium, that produces compactin. Microorganism is meant to include, e.g., microbial cells which are intact, immobilized or permeabilized. Production of compactin by the microorganism is meant to include the microorganism using its own natural gene or genes, or fragments thereof, to produce the compactin, and/or the microorganism using a foreign gene or genes, or fragments thereof, to produce the compactin. The foreign gene can be introduced into the microorganism by standard molecular cloning techniques known in the art, or by any other means which will result in expression of the compactin producing gene or genes, or fragments thereof, in the microorganism. In certain embodiments, the compactin is provided by using a cell free extract of a microorganism that produces compactin. The cell free extract can be prepared by a variety of methods known in the art, e.g., by physical or chemical means, so as to rupture the cells. Such methods include, e.g., grinding, ultrasonic treatment, or treatment with an enzyme or surface active agent. Preferably, the cell free extract is prepared with a French Press. Cell free extract is meant to include the material resulting from any of the preparation methods, or the soluble fraction resulting from any of the preparation methods. Preferably, the cell free extract comprises essentially the cellular contents without the particulate cellular debris, e.g., walls, e.g., wherein the walls have been essentially removed. Cell free extracts can be made from cells obtained before, during or after active growth of the cells. In other embodiments, the compactin can be provided by providing cell free culture media from a pregrown culture of a microorganism that produces compactin. In other embodiments, the compactin can be provided by providing a solution comprising compactin. In yet other embodiments, the compactin can be provided by using semi-purified or substantially purified compactin. The compactin can be free or immobilized. Any other source of compactin can be used in this invention. Compactin can be used in this invention at any concentration which will result in production of pravastatin. Preferably, the compactin concentration is between about 0.1 and about 100 g/liter, more preferably is between about 0.2 and about 25 g/liter, and most preferably is between about 1 and about 10 g/liter. In some embodiments, the compactin is added to the Actinomadura agent in shots. By shots is meant sequential additions of the agent. See Example 3.
The compactin is contacted with an agent derived from Actinomadura. Actinomadura is a genus belonging to the filamentous bacteria, actinomycetes. Actinomadura is meant to include wild type or any mutants which possess the ability to convert compactin to pravastatin. The mutants can be derived, e.g., spontaneously, or from physical agents, e.g., ultraviolet radiation, high frequency electromagnetic waves or nuclear radiation, or chemical agents, or from genetic engineering techniques.
Morphological and chemotaxonomic properties of a newly isolated strain included in this invention were compared with those of similar taxa and the results showed that the strain belongs to the genus Actinomadura. Actinomadura is a genus of aerobic actinomycetes. Actinomadura is a Gram positive, nonacid-fast, filamentous bacterium having branched, nonfragmenting filaments. Granules of Actinomadura are composed of branching, beaded, Gram positive filaments. The filaments are delicate. The growth rate of Actinomadura is slow. It grows on routine mycologic or mycobacteriologic media, under aerobic conditions. Deposit of these Actinomadura cells (strain 2966) has been made on Jun. 1, 1995, with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, and has been assigned deposit number ATCC 55678. Taxonomic properties of strain 2966 follow:
Description of strain 2966: Cell wall analysis: Whole cell hydrolysates contain meso-diaminopimelic acid (DAP) and madurose. Morphology: Strain 2966 produces an extensively branched, non-fragmenting and dense substrate mycelium. The aerial mycelium is very well developed on yeast malt extract agar, oatmeal agar, N-Z Amine with soluble starch and glucose agar and absent on ISP-4, ISP-5 and ISP-7 media. Spore chains noticeably exceed the diameter of the hyphae which is characteristic for Actinomadura strains and distinguish them from Streptomyces. The presence of meso-DAP is another distinguishing feature. Spores of strain 2966 have a round shape unlike glycomyces species. No special morphological structures were observed either on the substrate mycelium or aerial mycelium. The color of the aerial mycelium is white and yellow-brown for substrate mycelium. No soluble or melanin pigments are formed. Mycelium is not sensitive to pH changes. Carbon source utilization is very poor for glucose, arabinose, D-fructose, D-mannitol, raffinose, rhamnose, sucrose, xylose, galactose and there is only a trace of growth with i-inositol, salicin or on control agar. Strain 2966 shows antimicrobial activity against Micrococcus luteus and Bacillus subtilis, has resistance to penicillin and is inhibited by neomycin. Of the meso-DAP-containing actinomycetes, only Actinomadura species have non-fragmenting mycelium and display the same type of aerial mycelium formation and sporulation as strain 2966.
The agent derived from Actinomadura is meant to include, e.g., an enzyme, e.g., a hydroxylation enzyme, e.g., a hydroxylase, or any active portion thereof, that is able to convert compactin to pravastatin. The Actinomadura agent can be made in vivo or in vitro, e.g., in cell-free systems or by chemical synthesis. The agent includes wild type and mutated forms. The mutated agents can be derived spontaneously or from mutating the Actinomadura, e.g., with physical or chemical agents, or from utilizing genetic engineering techniques known to those skilled in the art. Mutated agents can also be synthesized by chemical techniques known to those skilled in the art. The agent is also meant to include, e.g., an agent derived from a genetically engineered strain of Actinomadura in which the nucleic acid coding for the agent or any active portion thereof, is operatively connected to a regulatory region, or portion thereof, that is different from the wild type regulatory region for the agent. In certain embodiments, the nucleic acid coding for the Actinomadura agent is cloned into another type of cell, e.g., a bacterium, e.g., Escherichia coli, Bacillus subtilis, Bacillus brevis or Streptomyces lividans; a fungus, e.g., Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha; insect cells; transgenic plants; or transgenic animals. In a preferred embodiment, the Actinomadura agent is cloned into a microorganism which is able to produce compactin, e.g., with the microorganism""s own gene(s), or with foreign gene(s).
The agent of this invention is a constitutive enzyme, and therefore does not require induction with compactin. See Example 6. Mg++, ATP and ascorbic acid, alone or in combination, but not Fe++ or Fe+++, stimulates the enzymatic activity of the agent. See Example 8. Preferrably, NADPH is used as a H+ donor, though NADH or other H+ donors can be used. Cofactors, e.g., xe2x88x9d-ketoglutarate, CoCl2, NiCl2, CuSo4, FMN or FAD, do not stimulate enzymatic activity of the agent. The agent is cytochrome P450 system-independent. See Example 7.
The compactin can be contacted with the agent in any way which enables the agent to convert the compactin to pravastatin. For example, the compactin can be contacted with whole cells of Actinomadura, or with a cell free extract of Actinomadura, or with cell free culture media from a pregrown culture of Actinomadura, or with a solution comprising the Actinomadura agent, or with semi-purified or substantially purified Actinomadura agent.
In certain embodiments, a pregrown culture of a microorganism that produces compactin is contacted with a pregrown culture of Actinomadura. By pregrown culture is meant a culture which has been inoculated with cells and cultured for some period of time, preferably about 1 to about 8 days, more preferably about 1 to about 5 days, and most preferablby about 2 to about 4 days. In other embodiments, a pregrown culture of a microorganism is contacted with a starting culture of Actinomadura. By starting culture is meant a culture which is inoculated with cells from, e.g., a slant culture, a frozen culture, a lyophilized culture or a liquid seed culture. In other embodiments, a starting culture of, a microorganism that produces compactin is contacted with a pregrown culture of Actinomadura. In other embodiments, a starting culture of a microorganism that produces compactin is contacted with a starting culture of Actinomadura. This mixture of cultures is grown for some period of time, preferably about 0.5 to about 8 days, more preferably about 1 to about 5 days, and most preferably about 2 to about 4 days.
In other embodiments, a cell free extract of a microorganism that produces compactin is contacted with a culture, e.g., starting or pregrown, of Actinomadura. In other embodiments, a culture, e.g., starting or pregrown, of a microorganism that produces compactin is contacted with a cell free extract of Actinomadura. And, in yet other embodiments, a cell free extract of a microorganism that produces compactin is contacted with a cell free extract of Actinomadura. In other embodiments, a culture, e.g., starting or pregrown, of a microorganism that produces compactin is contacted with a cell free extract of Actinomadura. Combinations using semi-purified or substantially purified compactin, or semi-purified or substantially purified Actinomadura agent are also included in this invention.
The conversion reaction of compactin to pravastatin can be done using any conditions which will result in the production of pravastatin. Any method of cultivation can be used, e.g., fermentation techniques, e.g., batch culture, fed-batch culture, continuous or solid-state culture. Preferably, an agitated liquid submerged culture is used, most preferably of the type useful for large scale industrial fermentation. Additives can be used during growth which contribute to the development of the hydroxylating system within the cells.
The preferred temperature is about 18xc2x0 C. to about 50xc2x0 C., more preferably about 25xc2x0 C. to about 37xc2x0 C., and most preferably about 28xc2x0 C. to about 31xc2x0 C. The preferred pH is about 5 to about 10, more preferably about 6 to about 8.5, and most preferably about 7.2 to about 8.0. Preferably, aerobiosis is provided, e.g., by agitation and/or aeration. The preferred shaking condition is about 0 rpm to about 400 rpm, more preferably about 200 rpm to about 250 rpm, and most preferably about 220 rpm.
The invention is meant to cover any percentage of conversion of compactin to pravastatin by the Actinomadura agent, preferably at least about 10%, more preferably at least about 25%, more preferably yet at least about 40%, more preferably yet at least about 50%, more preferably yet at least about 60%, more preferably yet at least about 70%, and most preferably at least about 80%. The percentage conversion is calculated by dividing the concentration of pravastatin by the concentration of compactin charged (initially added) and multiplying by 100.
In preferred embodiments, the pravastatin is isolated. Isolated is meant to include, e.g., enriched, separated or purified. Isolation can be by any method known to those skilled in the art, including, e.g., precipitation; extraction, e.g., with a solvent, e.g., ethyl acetate or butanol, and removal of the solvent, e.g., by distillation; chromatography, e.g., thin layer chromatography or column chromatography, e.g., using a matrix, e.g., alumina or silica gel, followed by elution. A preferred chromatographic method includes, e.g., high pressure liquid chromatography (HPLC). See, e.g., Serizawa et al., J. Antibiotics (1983).
The invention also includes a cell free extract from Actinomadura that has an agent, e.g., a hydroxylase, that converts compactin to pravastatin. The cell free extract can be obtained by any method, including those described above. Preferably, the cell free extract comprises essentially the cellular contents without particulate cellular debris, e.g., walls, e.g., wherein the walls have been essentially removed. Preferably, conversion of compactin to pravastatin by the cell free extract is at least about 10%, more preferably at least about 25%, more preferably yet at least about 40%, more preferably yet at least about 50%, more preferably yet at least about 60%, more preferably yet at least about 70%, and most preferably at least about 80%.
The invention also includes a hydroxylase, e.g., semi-purified or substantially purified, from Actinomadura that converts compactin to pravastatin. The hydroxylase is a constitutive enzyme. The activity of the hydroxylase is stimulated by any of ATP, ascorbic acid and Mg++, or by combinations thereof, but not by Fe++ or Fe+++. Preferably, NADPH is used as a H+ donor, though NADH or other H+ donors can be used. Cofactors, e.g., xe2x88x9d-ketoglutarate, CoCl2, NiCl2, CuSo4, FMN or FAD, do not stimulate the activity of the hydroxylase. The hydroxylase is cytochrome P450 system-independent.
The invention also includes Actinomadura ATCC 55678, having an agent, e.g., a hydroxylase, for converting compactin to pravastatin.
The invention further includes a method for treating a mammal to lower the blood cholesterol level of the mammal. Pravastatin, derived from compactin by contacting the compactin with an agent, e.g., a hydroxylase, derived from Actinomadura that converts compactin to pravastatin, is provided. The pravastatin is administered to a mammal in need of such treatment to cause a lower blood cholesterol level in the mammal.
By mammal is meant human as well as non-human mammals. Treating a mammal to lower blood cholesterol levels is meant to include, e.g., preventing or lowering high blood cholesterol levels.
Administration of the pravastatin can be accomplished by any method which allows the pravastatin to reach its target. By target is meant the place where the pravastatin is able to inhibit the cholesterol biosynthetic pathway in the mammal. The administration methods include, e.g., injection, deposition, implantation, suppositories, oral ingestion, inhalation, topical administration, or any other method of administration where access to the target by pravastatin is obtained. Injections can be, e.g., intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal. Implantation includes inserting implantable drug delivery systems, e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, polymeric systems, e.g., matrix erosion and/or diffusion systems and non-polymeric systems, e.g., compressed, fused or partially fused pellets. Suppositories include glycerin suppositories. Oral ingestion doses, e.g., pills, can be enterically coated. Inhalation includes administering pravastatin with an aerosol in an inhalator, either alone or attached to a carrier that can be absorbed.
The pravastatin can be suspended in a liquid, e.g., in dissolved form or colloidal form. The liquid can be a solvent, partial solvent or non-solvent. In many cases, water or an organic liquid can be used. Administration of pravastatin can be alone or in combination with other therapeutic agents. In certain embodiments, the pravastatin can be combined with a suitable carrier, e.g., a pharmaceutically acceptable carrier. In certain embodiments, the pravastatin is incorporated into a liposome or incorporated into a polymer release system.
In certain embodiments of the invention, the administration can be designed so as to result in sequential exposures to the pravastatin over some time period, e.g., hours, days, weeks, months or years. This can be accomplished by repeated administrations of the pravastatin by one of the methods described above, or alternatively, by a controlled release delivery system in which the pravastatin is delivered to the mammal over a prolonged period without repeated administrations. Administration of such a system can be, e.g, by long acting oral dosage forms, bolus injections, transdermal patches and sub-cutaneous implants.
The pravastatin can be administered prior to or subsequent to the appearance of high blood cholesterol levels. In certain embodiments, the pravastatin is administered to subjects who have a family history of high blood cholesterol levels, or who have a genetic predisposition for this condition. In other embodiments, the pravastatin is administered to subjects who have reached a particular age and who, therefore, are more likely to be affected by high blood cholesterol levels. In yet other embodiments, the pravastatin is administered to subjects who exhibit either early or advanced symptoms of the condition. The pravastatin can also be administered as a preventive measure.
The pravastatin is administered to the mammal in a therapeutically effective amount. By therapeutically effective amount is meant that amount which is capable of at least partially preventing or reversing high blood cholesterol levels. A therapeutically effective amount can be determined on an individual basis and will be based, at least in part, on consideration of the species of mammal, the mammal""s size, the type of delivery system used, the time of administration relative to the level of blood cholesterol, and whether a single, multiple, or controlled release dose regimen is employed. A therapeutically effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
Preferably, the dosage of pravastatin for a human is about 0.1 to about 5000 mg/day, more preferably it is about 1 to about 500 mg/day, and most preferably it is about 10 to about 50 mg/day. Preferably, the dosage form is such that it does not substantially deleteriously affect the mammal.